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Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

Shen K, Ramirez B, Mapes B, Shen GR, Gokhale V, Brown ME, Santarsiero B, Ishii Y, Dudek SM, Wang T, Garcia JG - PLoS ONE (2015)

Bottom Line: Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs.Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies.These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

View Article: PubMed Central - PubMed

Affiliation: Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

No MeSH data available.


Related in: MedlinePlus

Local conformational changes in the 31-253aa segment of nmMLCK1 [template: titin Z1Z2 (PDB ID: 1ya5A)] upon S147P SNP mutation and energy minimization.(A) The S147P SNP site localized in a potentially flexible loop connecting the 1st IGc2 domain and the 2nd IGc2 domain (with side chains of some neighboring residues shown in ball and stick model). (B) to (D), a closer look at the loop connecting the 1st IGc2 domain and the 2nd IGc2 domain after energy minimization (131-154aa shown in ball and stick model): (B) the loop in the wild type; (C) the loop in the P147 SNP variant; (D) overlay of the two loops (structural changes caused by the P147 SNP mutation shown in pink).
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pone.0130515.g005: Local conformational changes in the 31-253aa segment of nmMLCK1 [template: titin Z1Z2 (PDB ID: 1ya5A)] upon S147P SNP mutation and energy minimization.(A) The S147P SNP site localized in a potentially flexible loop connecting the 1st IGc2 domain and the 2nd IGc2 domain (with side chains of some neighboring residues shown in ball and stick model). (B) to (D), a closer look at the loop connecting the 1st IGc2 domain and the 2nd IGc2 domain after energy minimization (131-154aa shown in ball and stick model): (B) the loop in the wild type; (C) the loop in the P147 SNP variant; (D) overlay of the two loops (structural changes caused by the P147 SNP mutation shown in pink).

Mentions: By examining NMR and cell signaling results, we observed differential cell signaling between wild type and SNP mutants of nmMLCK1 (unpublished data) but only minor structural differences in their N-termini (by NMR). Since there are no available structures for these variants, we utilized bioinformatic and homology modeling tools to study them. In homology modeling using Swiss-Model [31], we first identified titin Z1Z2 domains (PDB ID: 1ya5A) [40] as an optimal template (sequence identity, 28.57%; QMEAN Z-score, –3.39) [32] for modeling the segment 31-253aa of nmMLCK1 that spans the first two IGc2 domains and their linker region (Fig 5A). Leveraging the HSQC results obtained, we hypothesized the P147 SNP variant to produce greater structural perturbations than other two SNP variants. Indeed, similar to NMR observation, the S147P SNP mutation induced only minor local conformational changes within the seemingly flexible loop that embraces the mutation (Fig 5B–5D). The root-mean-square deviation (RMSD) is 0.04 angstrom for the backbones of the linkage loops (137-149aa), and more reduced (0.01 angstrom) for the 31-253aa segments [33].


Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

Shen K, Ramirez B, Mapes B, Shen GR, Gokhale V, Brown ME, Santarsiero B, Ishii Y, Dudek SM, Wang T, Garcia JG - PLoS ONE (2015)

Local conformational changes in the 31-253aa segment of nmMLCK1 [template: titin Z1Z2 (PDB ID: 1ya5A)] upon S147P SNP mutation and energy minimization.(A) The S147P SNP site localized in a potentially flexible loop connecting the 1st IGc2 domain and the 2nd IGc2 domain (with side chains of some neighboring residues shown in ball and stick model). (B) to (D), a closer look at the loop connecting the 1st IGc2 domain and the 2nd IGc2 domain after energy minimization (131-154aa shown in ball and stick model): (B) the loop in the wild type; (C) the loop in the P147 SNP variant; (D) overlay of the two loops (structural changes caused by the P147 SNP mutation shown in pink).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482139&req=5

pone.0130515.g005: Local conformational changes in the 31-253aa segment of nmMLCK1 [template: titin Z1Z2 (PDB ID: 1ya5A)] upon S147P SNP mutation and energy minimization.(A) The S147P SNP site localized in a potentially flexible loop connecting the 1st IGc2 domain and the 2nd IGc2 domain (with side chains of some neighboring residues shown in ball and stick model). (B) to (D), a closer look at the loop connecting the 1st IGc2 domain and the 2nd IGc2 domain after energy minimization (131-154aa shown in ball and stick model): (B) the loop in the wild type; (C) the loop in the P147 SNP variant; (D) overlay of the two loops (structural changes caused by the P147 SNP mutation shown in pink).
Mentions: By examining NMR and cell signaling results, we observed differential cell signaling between wild type and SNP mutants of nmMLCK1 (unpublished data) but only minor structural differences in their N-termini (by NMR). Since there are no available structures for these variants, we utilized bioinformatic and homology modeling tools to study them. In homology modeling using Swiss-Model [31], we first identified titin Z1Z2 domains (PDB ID: 1ya5A) [40] as an optimal template (sequence identity, 28.57%; QMEAN Z-score, –3.39) [32] for modeling the segment 31-253aa of nmMLCK1 that spans the first two IGc2 domains and their linker region (Fig 5A). Leveraging the HSQC results obtained, we hypothesized the P147 SNP variant to produce greater structural perturbations than other two SNP variants. Indeed, similar to NMR observation, the S147P SNP mutation induced only minor local conformational changes within the seemingly flexible loop that embraces the mutation (Fig 5B–5D). The root-mean-square deviation (RMSD) is 0.04 angstrom for the backbones of the linkage loops (137-149aa), and more reduced (0.01 angstrom) for the 31-253aa segments [33].

Bottom Line: Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs.Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies.These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

View Article: PubMed Central - PubMed

Affiliation: Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

No MeSH data available.


Related in: MedlinePlus